The following description provides a summary of information relevant to the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to the invention.
The immobilization of oligonucleotides on substrates is an important and necessary step for many applications such as DNA chip technology, surface plasmon resonance experiments, or other biosensor applications. Classically, oligonucleotides are immobilized onto substrates by modification of the 3′- or 5′-end with one reactive group e.g. an amine, thiol or aldehyde (covalent attachment) or group forming stable complexes e.g. biotin, phenylboronic acid etc. (noncovalent attachment). The modified oligonucleotides are then addressed to the location where the immobilization is desired and reacted with an appropriate functional group such as an aldehyde, maleimide, hydrazide etc. or complexed with a binding molecule such as streptavidin, etc. The addressing to specific locations on a substrate can be done by spotting (pin or drop deposition), by electronic addressing, or by a variety of other processes. In some cases the reaction for the immobilization is slow and requires long (overnight) incubation of the oligonucleotides on the substrate. These immobilization reactions may also be reversible, resulting in the release of the biomolecule over time.
In other instances, dendrimeric structures on biomolecules has been described (e.g., WO 99/10362, WO 96/19240, and WO 99/43287), but the use of the dendrimeric structures have been directed toward providing signal sites such as for detection while the biomolecule itself is simply attached to a substrate using classical means.
In contrast thereto, the present invention describes an improved process for immobilization of biomolecules using oligonucleotides containing multiple reactive sites, i,e. nucleophiles, electrophiles, and Lewis acids or bases. The advantage of this approach is a higher rate of immobilization, a higher stability of the attachment, and the potential to obtain higher amounts of immobilized oligonucleotide onto the substrate surface. These gains are independent of the approach used for the immobilization. Oligonucleotides with multiple attachment sites can be obtained with both covalent and noncovalent attachment chemistries.
Furthermore the present invention describes the preparation of oligonucleotides containing one or more hydrazides. Hydrazides are nucleophilic reactive groups that can be nsed for any type of conjugation reaction. They can react, for example, with electrophilie aldehydes forming hydrazones (which can be further stabilized by reduction) and with active esters forming stable covalent linkages, see FIG. 18. This chemistry can be used for attaching fluorophores, proteins or peptides, reporter groups and other oligomers to oligonucleotides. The reactions of hydrazides can also be used for the immobilization of biomolecules onto substrates. Such hydrazide modified oligonucleotides have not been previously described.
The advantages of this invention within the scope of this description are numerous. For example, this invention uses a short reaction time, allows for multiple binding sites per bound entity, provides for stability to a relatively broad pH range, and provides for the capability of attachment under both anhydrous or aqueous conditions thereby providing an, improved method for attaching molecules to any solid phase surface for any applicable use. The invention is useful for solid phase synthesis and/or synthesis of small molecule libraries such as biomolecules including, but not limited to, DNA, RNA, PNA, p-RNA (pyranosyl-RNA), and peptides. The invention is also useful for analytical techniques that require an immobilized reagent such as, without limitation, hybridization based assays, diagnostics, gene sequence identification and the like.